Required Practical Activity 4

Investigate the effect of pH on the rate of amylase enzyme

Teacher and technician Sheet

Students should use a continuous sampling technique to determine the time taken to
completely digest a starch solution at a range of pH values. Iodine reagent is to be used to test
for starch every x seconds. Temperature must be controlled by use of a water bath or electric
heater.

Suggested apparatus
1% Amylase enzyme
1% Starch solution
iodine solution (harmful)
x5 pH buffer solutions
universal indicator (used to check pH of solution)
thermostatically controlled water bath
stopwatch
test tubes x5 per group
measuring cylinders (10 cm3)
dropper pipettes
syringes x2 5 cm3 (1 for amylase and 1 for starch solution)
Test tube rack
Marker pens

Apparatus and Techniques

AT 1 – use appropriate apparatus to record the volumes of liquids, time and pH.
AT 2 – safe use of a water bath or electric heater.
AT 5 – measure the rate of reaction by the colour change of iodine indicator

Suggested method

PREPARATION NOTES
Check the speed of the reaction with the suggested volumes of reactants to be used – 2 cm3 of
starch: 2 cm3 of amylase: 1 cm3 of buffer at pH 6. Ideally the reaction should take about 60
seconds at this pH: this is the usual optimum for amylase

PORCEDURE
1. Place single drops of iodine solution in rows on the tile.

2. Label a test tube with the pH to be tested.

3. Use the syringe to place 2 cm3 of amylase into the test tubes.
Required Practical Activity 4
4. Add 1 cm3 of buffer solution to the test tube using a syringe.
5. Place the test tube into a water bath set at 37 degrees and leave for 5 minutes
6. Use another syringe to add 2 cm3 of starch solution to the amylase/ buffer solution, start the
stop clock and leave it on throughout the test. Mix using a plastic pipette.
7. After 10 seconds, use the plastic pipette to place one drop of the mixture on the first drop of
iodine. The iodine solution should turn blue-black. If the iodine solution remains orange the
reaction is going too fast and the starch has already been broken down. Squirt the rest of the
solution in the pipette back into the test tube.
8. Wait another 10 seconds. Then remove a second drop of the mixture to add to the next drop of
iodine.
9. Repeat step 8 until the iodine solution and the amylase/ buffer/ starch mixture remain orange.
10. You could prepare a control drop for comparison with the test drops. What should this contain?
11. Count how many iodine drops you have used, each one equalling 10 seconds of reaction time.
12. Repeat the whole procedure with another of the pH buffers to be used, or pool the class
results.
13. Consider collecting repeat data if there is time.
14. Plot a graph of time taken to break down starch against pH, or calculate the rate of reaction
and plot rate against pH.

Variables
Independent – pH of starch solution
Dependent – time taken for reacting mixture to turn black
Controlled variable – temperature, intervals of testing, amount of starch/amylase/pH buffer

Suggested results table

Time taken for colour change

pH Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Average

Risk assessment

Risk Hazards Control Measure

Possible mathematical tests

Rates, gradients, means, standard deviation,